Interferons are proteins which are produced by a number of different kinds of organisms and which are presently grouped into three major classes, designated leukocyte interferon (IFN-.alpha.), fibroblast interferon (IFN-.beta.) and immune interferon (IFN-.gamma.). The interferons have antiviral and antiproliferative activities, a potent ability to confer a virus-resistant state in targeted cells and immunomodulatory activities. Their biological properties have led to the clinical use of interferons as therapeutic agents for the treatment of viral infections and malignancies.
Interferons have been produced from natural sources such as buffy coat leukocytes and fibroblast cells, optionally using known inducing agents to increase the production of interferon. Interferons have also been produced by recombinant DNA techniques, i.e. by expression from a microorganism which has been transformed with an expression vector containing an inteferon gene under the control of a promoter-operator sequence. (Leukocyte, fibroblast and immune interferons produced by recombinant techniques are designated rIFN-.alpha., rIFN-.beta. and rIFN-.gamma., respectively). As many as 12 distinct genes encoding for different species of rIFN-.alpha. have been cloned. These various species are designated rIFN-.alpha.A, rIFN-.alpha.B, rIFN-.alpha.C and so forth.
Goeddel and coworkers achieved the initial expression of rIFN-.alpha.A in E. coli cells containing the recombinant plasmid pL 31. (Nature, 287, 411 (1980)). This plasmid contains the structural gene for mature rIFN-.alpha.A (i.e., a gene in which the nucleotide sequence encoding a 23-amino acid signal peptide normally translated in the human cell has been removed and an ATG "start" signal has been inserted immediately before the codon for the first amino acid following the signal peptide) under the control of an appropriately positioned promoter-operator sequence. The rIFN-.alpha.A produced in this manner has been employed in the clinical treatment of patients suffering from a variety of viral and neoplastic diseases.
The rIFN-.alpha. interferons are 165 amino acids (in the case of rIFN-.alpha.A) or 166 amino acids in length, except that they may, in some instances contain a methionine attached to the N-terminus of the ordinarily first amino acid of the protein as the result of translation of the ATG start signal which encodes methionine.
Hybrid leukocyte interferons have been produced by expression of genes which are produced by cleaving two or more genes encoding different leukocyte interferons at internal endonuclease cleavage sites and then ligating one or more cleavage fragments of one gene with one or more cleavage fragments of a different gene (or genes) to produce a gene encoding a complete 165- or 166-amino acid leukocyte interferon having one or more segments corresponding to a portion of a first leukocyte interferon species and one or more segments corresponding to portions of different leukocyte interferon species. In this manner, for example, it has been possible to produce a leukocyte interferon in which the amino acid sequence corresponds to that of rIFN-.alpha.A at positions 1-91 and to that of rIFN-.alpha.D at positions 92-166. Similarly, by ligating the gene cleavage fragments in reverse order, it has been possible to produce a leukocyte interferon in which the amino acid sequence corresponds to that of rIFN-.alpha.D at positions 1-92 and to that of rIFN-.alpha.A at positions 93-165. (J. Biol. Chem., 257, pp. 11497-11502 ( 1982)).
A problem which has occurred in the manufacture and use of interferons is that the individual interferon molecules tend to oligomerize. The etiology of these oligomers has not been completely understood. It is believed, however, that the procedures used to purity interferons for therapeutic use may contribute to the oligomerization problem. Presently available purification methods, such as high pressure liquid chromatography or monoclonal antibody affinity chromatography are carried out under conditions which can favor the formation of dimers, trimers and higher oligomers of interferon. These oligomeric forms of interferon result from two or more interferon molecules becoming irreversibly associated with one another through intermolecular covalent bonding, such as by disulfide linkages. This problem has been observed particularly with respect to leukocyte and fibroblast interferons.
While the dimeric forms of interferons are believed to retain biological activity, the higher oligomeric forms in many cases have either no biological activity or significantly reduced activity by comparison to the monomeric forms. Moreover, the oligomeric forms have the potential for causing deleterious side effects if used therapeutically.
All of the known rIFN-.alpha., rIFN-.beta. and rIFN-.gamma. interferons contain multiple cysteine residues. These residues contain sulfhydryl side-chains which are capable of forming intermolecular disulfide bonds, which result in oligomerization, as well as intramolecular disulfide bonds. The amino acid sequence of rIFN-.alpha.A contains cysteine residues at positions 1, 29, 98 and 138. Wetzel and coworkers assigned intramolecular disulfide bonds between the cysteine residues and positions 1 and 98 and between the cysteine residues and positions 29 and 138. Nature, 289, 606 (1981).
Because of the importance of eliminating or preventing the occurrence of oligomers in interferon preparations, considerable efforts have been expended to overcome the oligomerization problem. Heretofore, efforts have been concentrated on adjusting the purification conditions to prevent the formation of oligomers or post-processing with reagents and reaction conditions which reduce intermolecular disulfide bonds.